Organs for transplantation

ABSTRACT

The invention provides methods and compositions that improve the success of organ transplantation. The methods and compositions are directed to exposing a desired organ to stem cells prior to, during, and/or after transplantation. In one embodiment, the stem cells reduce the deleterious effects of ischemia on an organ designated to be harvested for transplantation or that has been harvested for transplantation. In another embodiment in which an organ designated for transplantation is perfused ex vivo, the method involves reducing ischemic reperfusion injury by perfusing the organ with a medium that contains stem cells.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 8, 2014, isnamed ATH-022234USORD_SL.txt and is 7,517 bytes in size.

FIELD OF THE INVENTION

The field of the invention is organ transplantation and providingmethods and compositions that improve the success of organtransplantation. The methods and compositions are directed to exposing adesired organ, prior to or during transplantation, to stem cells. In oneembodiment, the stem cells reduce the deleterious effects of ischemia onan organ designated to be harvested for transplantation or that has beenharvested for transplantation. In another embodiment, the organ is alung. In a further embodiment in which an organ designated fortransplantation is perfused ex vivo, the method involves reducingischemic reperfusion injury by perfusing the organ with a medium thatcontains stem cells.

BACKGROUND OF THE INVENTION

Organ transplantation represents the preparation and harvesting of anorgan from a donor or a donor site (if the donor and recipient are thesame), and the implantation, maintenance and/or use of the organ into orby the recipient of the donated organ. It has been estimated that thereare more than 50,000 organ transplants performed per year in the majorhealthcare markets (e.g., U.S., Europe and Japan), and that there aremore than 170,000 patients on waiting lists for organ transplants.Demand for healthy organs significantly outstrips the supply.

A major challenge in organ transplantation has been transplantrejection, which can lead to significant complications in organ functionor to transplant failure. In general, this has been addressed throughthe matching of donors and recipients who have highly similar serotypes,and through the use of immunosuppressive drugs to manage theimmunological response underlying transplant rejection.

Another major challenge has been preservation of organ viability priorto and during the implantation procedure. The removal, storage andtransplantation of an organ may profoundly affect the internal structureand function of the organ and can influence significantly the degree towhich the return of normal organ function is delayed or prevented aftertransplantation is completed. Such organ injury occurs primarily as aresult of ischemia and hypothermia, but may also be related toreperfusion of the organ ex vivo or during implantation. Techniques fororgan preservation, including ex vivo perfusion, serve to minimize thisdamage to promote optimal graft survival and function. But, even withthese techniques, the organ health will decline is many cases, affectingtransplantation outcome, and in some cases, the decline is sosignificant that the donated organs are rejected prior totransplantation as non-viable.

A technology that addresses these important challenges in organtransplantation should have a substantial impact on patient quality oflife and survival, and on the treatment of the complications associatedwith transplantation.

SUMMARY OF THE INVENTION

The invention provides a method comprising transplanting an organ thathas been exposed to exogenous stem cells prior to, during, and/or aftertransplantation. Exposure to the stem cells can improve the probabilityof a successful organ transplantation. Accordingly, the invention isdirected to the following embodiments.

In one embodiment, the method may involve tolerizing the organ bycontacting the organ with exogenous stem cells prior to, during, and/orafter transplantation. By tolerizing the organ, that organ is betterprepared to be accepted by a recipient without significant immunologicalinterference. Tolerization can be achieved, for example, by theinduction of T-regulatory cells in the organ (see, e.g., Eggenhofer etal., Stem Cells Translation Medicine 2013; 2:000-000).

In one embodiment, the invention is directed to a method to reduceinjury in an organ ex vivo by contacting the organ with a medium thatcontains exogenous stem cells prior to, during, and/or aftertransplantation.

In one embodiment, the injury occurs as a result of ischemic reperfusioninjury.

In one embodiment, the method is directed to reducing general tissue orcell degradation in the organ to be transplanted. This may result fromfactors including, but not limited to, ischemia, hypothermia andreperfusion. Accordingly, in one embodiment, the invention is directedto reducing injury as a result of one or more of these events. Suchevents may be caused, at least in part, by one or a combination of thefollowing: (1) immunomodulation of TH1 T-cells to TH2 T-cells; (2)immunomodulation of M1 macrophages to M2 macrophages (e.g., causing ashift from a pro-inflammatory response to an anti-inflammatoryresponse); (3) inhibiting the infiltration of neutrophils (e.g., byreducing the cell surface receptors); (4) shifting neutrophils frombeing pro-inflammatory to anti-inflammatory; and (5) cytoprotection oranti-apoptotic effects generated by the exogenous stem cells.

The events described herein may result in inflammation, otherimmunological response, cytokine production, cell apoptosis, and otherevents that affect the viability of an organ and suitability fortransplantation. Accordingly, in one embodiment, the invention isdirected to reducing the deleterious effects of these events byadministering exogenous stem cells to an organ that is subject to theseevents or in which these events have already occurred.

Examples of events that may result in inflammation, other immunologicalresponse, cytokine production, cell apoptosis, and other events thataffect the viability of an organ and suitability for transplantation caninclude, but are not limited to, those associated with endothelialresponse, reactive oxygen species, complement, and leukocytes. Eventsassociated with endothelial response can include, but are not limitedto, expression of certain pro-inflammatory gene products (e.g.,leukocyte adhesion molecules, cytokines) and/or bioactive agents (e.g.,endothelin, thromboxane A₂) and/or repression of other “protective” geneproducts (e.g., constitutive nitric oxide synthase, thrombomodulin)and/or bioactive agents (e.g., prostacyclin, nitric oxide). Eventsassociated with reactive oxygen species (e.g., (O2-), (OH—), (HOCl),(H2O2), and nitric oxide-derived peroxynitrite) can include, but are notlimited to, direct damage to cellular membranes by lipid peroxidation,stimulating leukocyte activation and chemotaxis by activating plasmamembrane phospholipase A2 to form arachidonic acid (thromboxane A2 andleukotriene B4), and/or increasing leukocyte activation, chemotaxis, andleukocyte-endothelial adherence after ischemic reperfusion. Eventsassociated with complement activation, such as C3a, C5a, iC3b, C5b9 (C5ais most potent) can include, but are not limited to, formation ofseveral pro-inflammatory mediators that alter vascular homeostasis by,e.g., compromising blood flow to an ischemic organ by altering vascularhomeostasis and increasing leukocyte-endothelial adherence. Eventsassociated with leukocytes can include, but are not limited to,leukocyte activation, chemotaxis, leukocyte-endothelial cell adhesionand transmigration, which may further lead to mechanical obstruction, asleukocytes release toxic ROS, proteases, and elastases, resulting inincreased microvascular permeability, edema, thrombosis, and parenchymalcell death.

In one embodiment, the organ is selected from the group including, butnot limited to, lung, kidney, heart, liver, pancreas, thymus,gastrointestinal tract and composite allografts, such as limbs, facesand the like, and tissues including, but not limited to, cornea, skin,veins, arteries, bones, tendons and valves, such as heart valves and thelike.

In one embodiment, the stem cells reduce inflammation in the organ. Forexample, the organ can be exposed to the stem cells for a time and dosesufficient to reduce inflammation in the organ.

In one embodiment, the stem cells reduce the occurrence of inflammatorycells in the organ. For example, the organ can be exposed to the stemcells for a time and with a dose sufficient to reduce the occurrence ofinflammatory cells in the organ.

In one embodiment, the stem cells reduce inflammatory cytokines in theorgan. For example, the organ can be exposed to the stem cells for atime and with a dose sufficient to reduce inflammatory cytokines in theorgan.

In one embodiment, the stem cells reduce the occurrence of pulmonaryedema. For example, the organ can be exposed to the stem cells for atime and with a dose sufficient to reduce the occurrence of pulmonaryedema.

In one embodiment, the stem cells increase the occurrence of IL-10expression (protein and/or mRNA) in pulmonary tissue. For example, theorgan can be exposed to the stem cells for a time and with a dosesufficient to increase the occurrence of IL-10 expression in pulmonarytissue.

In one embodiment injury results from hypoxia in the organ.

In one embodiment, the stem cells reduce the effects of hypoxia in theorgan.

In one embodiment, the stem cells are administered at any time betweenremoval of the organ from the donor and transplantation into therecipient.

In one embodiment, the stem cells are exposed to the organ during thetransplantation procedure.

In one embodiment, the organ can be exposed to the stem cells while theorgan is still intact in the donor but before removal of the organ fromthe donor.

In one embodiment, the organ can be exposed to the stem cells for aperiod of time. The period of time can depend upon the particular organ.For example, the period of time can be about 1-2 hours, about 2-3 hours,about 3-4 hours, about 4-5 hours, about 5-6 hours, about 7-8 hours,about 8-9 hours, about 9-10 hours, or about 10 hours or more. Oneexample of a suitable period of time is described by Zhao et al., BMCMedicine 2012, 10:3, which is incorporated by reference herein for theteaching of an ex vivo procedure, including suitable time periods, foran intravenous ex vivo cell process.

In one embodiment, the concentration of stem cells exposed to the organcan depend upon the particular organ. For example, the concentration ofcells exposed to the organ can be about 0.01 to about 5×10⁷ cells/ml,about 1×10⁵ cells/ml to about 5×10⁷ cells/ml, or about 10×10⁶ cells/ml.

In another embodiment, the concentration of stem cells exposed to theorgan can be about 1×10⁵ cells/kg organ to about 5×10⁵ cells/kg organ,about 5×10⁵ cells/kg organ to 1×10⁶ cells/kg organ to 5×10⁶ cells/kgorgan, about 5×10⁶ cells/kg organ to 1×10⁷ cells/kg organ, about 1×10⁷cells/kg organ to 1.5×10⁷ cells/kg organ, or about 1×10⁷ cells/kg organto 2×10⁸ cells/kg organ.

In other embodiments, the stem cells are contained in a fluid forperfusion into the organ or in a carrier for intra-organ (such asintra-bronchially) administration.

In another embodiment, the stem cells are contained in a medium in whichthe organ is contacted prior to transplantation, such as a medium inwhich the organ is bathed rather than being perfused.

The inventors contemplate using any desired stem cell in the methods ofthe invention. These include, but are not limited to, embryonic stemcells, non-embryonic multipotent stem cells, mesenchymal stem cells,neural stem cells, induced pluripotent stem cells, and the like. In oneembodiment, the stem cells can be non-HLA matched, allogeneic cells.

Cells include, but are not limited to, cells that are not embryonic stemcells and not germ cells, having some characteristics of embryonic stemcells, but being derived from non-embryonic tissue, and providing theeffects described in this application. The cells may naturally achievethese effects (i.e., not genetically or pharmaceutically modified).However, natural expressors can be genetically or pharmaceuticallymodified to increase potency.

The cells may express pluripotency markers, such as oct4. They may alsoexpress markers associated with extended replicative capacity, such astelomerase. Other characteristics of pluripotency can include theability to differentiate into cell types of more than one germ layer,such as two or three of ectodermal, endodermal, and mesodermal embryonicgerm layers. Such cells may or may not be immortalized or transformed inculture. The cells may be highly expanded without being transformed andalso maintain a normal karyotype. In one embodiment, the non-embryonicstem, non-germ cells may have undergone a desired number of celldoublings in culture. For example, non-embryonic stem, non-germ cellsmay have undergone at least 10-40 cell doublings in culture, such as30-35 cell doublings, wherein the cells are not transformed and have anormal karyotype. The cells may differentiate into at least one celltype of each of two of the endodermal, ectodermal, and mesodermalembryonic lineages and may include differentiation into all three.Further, the cells may not be tumorigenic, such as not producingteratomas. If cells are transformed or tumorigenic, and it is desirableto use them for infusion, such cells may be disabled so they cannot formtumors in vivo, as by treatment that prevents cell proliferation intotumors. Such treatments are well known in the art.

Cells include, but are not limited to, the following numberedembodiments:

1. Isolated expanded non-embryonic stem, non-germ cells, the cellshaving undergone at least 10-40 cell doublings in culture, wherein thecells express oct4, are not transformed, and have a normal karyotype.

2. The non-embryonic stem, non-germ cells of 1 above that furtherexpress one or more of telomerase, rex-1, rox-1, or sox-2.

3. The non-embryonic stem, non-germ cells of 1 above that candifferentiate into at least one cell type of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages.

4. The non-embryonic stem, non-germ cells of 3 above that furtherexpress one or more of telomerase, rex-1, rox-1, or sox-2.

5. The non-embryonic stem, non-germ cells of 3 above that candifferentiate into at least one cell type of each of the endodermal,ectodermal, and mesodermal embryonic lineages.

6. The non-embryonic stem, non-germ cells of 5 above that furtherexpress one or more of telomerase, rex-1, rox-1, or sox-2.

7. Isolated expanded non-embryonic stem, non-germ cells that areobtained by culture of non-embryonic, non-germ tissue, the cells havingundergone at least 40 cell doublings in culture, wherein the cells arenot transformed and have a normal karyotype.

8. The non-embryonic stem, non-germ cells of 7 above that express one ormore of oct4, telomerase, rex-1, rox-1, or sox-2.

9. The non-embryonic stem, non-germ cells of 7 above that candifferentiate into at least one cell type of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages.

10. The non-embryonic stem, non-germ cells of 9 above that express oneor more of oct4, telomerase, rex-1, rox-1, or sox-2.

11. The non-embryonic stem, non-germ cells of 9 above that candifferentiate into at least one cell type of each of the endodermal,ectodermal, and mesodermal embryonic lineages.

12. The non-embryonic stem, non-germ cells of 11 above that express oneor more of oct4, telomerase, rex-1, rox-1, or sox-2.

13. Isolated expanded non-embryonic stem, non-germ cells, the cellshaving undergone at least 10-40 cell doublings in culture, wherein thecells express telomerase, are not transformed, and have a normalkaryotype.

14. The non-embryonic stem, non-germ cells of 13 above that furtherexpress one or more of oct4, rex-1, rox-1, or sox-2.

15. The non-embryonic stem, non-germ cells of 13 above that candifferentiate into at least one cell type of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages.

16. The non-embryonic stem, non-germ cells of 15 above that furtherexpress one or more of oct4, rex-1, rox-1, or sox-2.

17. The non-embryonic stem, non-germ cells of 15 above that candifferentiate into at least one cell type of each of the endodermal,ectodermal, and mesodermal embryonic lineages.

18. The non-embryonic stem, non-germ cells of 17 above that furtherexpress one or more of oct4, rex-1, rox-1, or sox-2.

19. Isolated expanded non-embryonic stem, non-germ cells that candifferentiate into at least one cell type of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages, said cellshaving undergone at least 10-40 cell doublings in culture.

20. The non-embryonic stem, non-germ cells of 19 above that express oneor more of oct4, telomerase, rex-1, rox-1, or sox-2.

21. The non-embryonic stem, non-germ cells of 19 above that candifferentiate into at least one cell type of each of the endodermal,ectodermal, and mesodermal embryonic lineages.

22. The non-embryonic stem, non-germ cells of 21 above that express oneor more of oct4, telomerase, rex-1, rox-1, or sox-2.

In one embodiment, a conditioned medium is used instead of the stemcells.

In one embodiment, the organ is from a human.

In view of the property of the cells to achieve the desired effects,cell banks can be established containing cells that are selected forhaving a desired potency (level of ability) to achieve the effects.Accordingly, the invention encompasses assaying cells for the ability.The bank can provide a source for making a pharmaceutical composition toadminister to an organ. Cells can be used directly from the bank orexpanded prior to use. Especially in the case that the cells aresubjected to further expansion, after expansion it is desirable tovalidate that the cells still have the desired potency. Banks allow the“off the shelf” use of cells that are allogeneic to the organ donor andrecipient.

Accordingly, the invention also is directed to diagnostic proceduresconducted prior to exposing the stem cells to an organ. The proceduresinclude assessing the potency of the cells to achieve the effectsdescribed in this application. The cells may be taken from a cell bankand used directly or expanded prior to administration. In either case,the cells could be assessed for the desired potency. Especially in thecase that the cells are subjected to further expansion, after expansionit is desirable to validate that the cells still have the desiredpotency.

Although the cells selected for the effects are necessarily assayedduring the selection procedure, it may be preferable, and prudent, toagain assay the cells prior to administration to a subject for treatmentto confirm that the cells still achieve the effects at desired levels.This is particularly preferable where the cells have been stored for anylength of time, such as in a cell bank, where cells are, most likely,frozen during storage.

Between the original isolation of the cells and the administration to anorgan, there may be multiple (i.e., sequential) assays for the effects.This is to confirm that the cells can still achieve the effects, atdesired levels, after manipulations that occur within this time frame.For example, an assay may be performed after each expansion of thecells. If cells are stored in a cell bank, they may be assayed afterbeing released from storage. If they are frozen, they may be assayedafter thawing. If the cells from a cell bank are expanded, they may beassayed after expansion. Preferably, a portion of the final cell product(that is physically administered to the organ) may be assayed.

Since the stem cells may provide the effects described herein by meansof secreted molecules, the various embodiments described herein foradministration of stem cells may be done by administration of one ormore of the secreted molecules, such as might be in conditioned culturemedium.

The invention is also directed to compositions comprising a populationof the cells having a desired potency to achieve the desired effects.Such populations may be found as pharmaceutical compositions suitablefor administration to an organ and/or in cell banks from which cells canbe used directly for administration or expanded prior to administration.In one embodiment, the cells have enhanced (increased) potency comparedto the previous (parent) cell population. Parent cells are as definedherein. Enhancement can be by selection of natural expressors or byexternal factors acting on the cells.

The cells may be prepared by the isolation and culture conditionsdescribed herein. In a specific embodiment, they are prepared by cultureconditions that are described herein involving lower oxygenconcentrations combined with higher serum, such as those used to preparethe cells designated “MultiStem®.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic of study design.

FIG. 2. Representative gross appearances of the right lower lobe (RLL)and left lower lobe (LLL) of lung 2 following reperfusion. TheMSC-treated LLL appears normal while the vehicle-treated RLL appearsedematous and inflamed.

FIG. 3. Semi-quantitative scoring demonstrates significant decrease inoverall inflammation in the MSC-treated LLL compared to thevehicle-treated RLL in 4 out of 5 lungs and in aggregate. Means±SD ofpooled observations from 3 blinded observers are depicted.

FIGS. 4A-B. Representative photomicrographs from lung 1 demonstrateminimal to no significant inflammation in MSC-treated LLL vs. alveolarseptal thickening, edema, and perivascular (FIG. 4A) and peri-bronchialinflammatory cell infiltrates (FIG. 4B). Original Mag 200×.

FIGS. 5A-C. Decrease in total BAL fluid cell counts in the MSC-treatedLLL in lungs 3-5 (FIG. 5A). Total cell counts were not assessed in lungs1 or 2. MSC instillation also resulted in a significant decreased in theelevated numbers of BAL fluid total neutrophils and eosinophils in all 5lungs (FIGS. 5B-C). Data represents means±SEM of pooled observationsfrom 3 blinded observers.

FIG. 6. Representative BAL fluid cytokine analyses from Lung 4demonstrate significant increase in IL-10 in the MSC-treated LLL but nosignificant change in iNOS, STC-1, or TSG-6. Data representsmeans+standard deviations from triplicate determinations of each LLL orRLL sample.

FIG. 7. Cytokine analysis of lung tissue. qPCR analysis was performed onthe lung tissue samples collected from the LLL and RLL of lungs 2-5 att=0, 2 and/or 4 hours. The fold-expression represents the levels of thetarget gene compared to the t=0 value. All data were normalized to ahousekeeping gene, GAPDH. Data represents means+standard deviations fromLLL and RLL samples from lungs 2-5.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand, as such, may vary. The terminology used herein is for the purposeof describing particular embodiments only, and is not intended to limitthe scope of the disclosed invention, which is defined solely by theclaims.

The section headings are used herein for organizational purposes onlyand are not to be construed as in any way limiting the subject matterdescribed.

The methods and techniques of the present application are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990).

Definitions

“A” or “an” means herein one or more than one; at least one. Where theplural form is used herein, it generally includes the singular.

A “cell bank” is industry nomenclature for cells that have been grownand stored for future use. Cells may be stored in aliquots. They can beused directly out of storage or may be expanded after storage. This is aconvenience so that there are “off the shelf” cells available foradministration. The cells may already be stored in apharmaceutically-acceptable excipient so they may be directlyadministered or they may be mixed with an appropriate excipient whenthey are released from storage. Cells may be frozen or otherwise storedin a form to preserve viability. In one embodiment of the invention,cell banks are created in which the cells have been selected forenhanced potency to achieve the effects described in this application.Following release from storage, and prior to administration, it may bepreferable to again assay the cells for potency. This can be done usingany of the assays, direct or indirect, described in this application orotherwise known in the art. Then cells having the desired potency canthen be administered. Banks can be made using autologous cells (derivedfrom the organ donor or recipient). Or banks can contain cells forallogeneic uses.

“Co-administer” means to administer in conjunction with one another,together, coordinately, including simultaneous or sequentialadministration of two or more agents.

“Comprising” means, without other limitation, including the referent,necessarily, without any qualification or exclusion on what else may beincluded. For example, “a composition comprising x and y” encompassesany composition that contains x and y, no matter what other componentsmay be present in the composition. Likewise, “a method comprising thestep of x” encompasses any method in which x is carried out, whether xis the only step in the method or it is only one of the steps, no matterhow many other steps there may be and no matter how simple or complex xis in comparison to them. “Comprised of and similar phrases using wordsof the root “comprise” are used herein as synonyms of “comprising” andhave the same meaning.

“Comprised of” is a synonym of “comprising” (see above).

The term “contact”, when used in relation to a stem cell and an organ tobe transplanted, can mean that, upon exposure to the organ, the stemcell physically touches the organ. In such instances, the stem cell isin direct contact with the organ. In other instances, the stem cell canindirectly contact the organ where one or more structures (e.g., anothercell) and/or fluids (e.g., blood) physically intervene(s) between thestem cell and the organ.

“EC cells” were discovered from analysis of a type of cancer called ateratocarcinoma. In 1964, researchers noted that a single cell interatocarcinomas could be isolated and remain undifferentiated inculture. This type of stem cell became known as an embryonic carcinomacell (EC cell).

“Effective amount” generally means an amount which provides the desiredlocal or systemic effect, e.g., effective to ameliorate undesirableeffects of inflammation, including achieving the specific desiredeffects described in this application. For example, an effective amountis an amount sufficient to effectuate a beneficial or desired clinicalresult. The effective amounts can be provided all at once in a singleadministration or in fractional amounts that provide the effectiveamount in several administrations. The precise determination of whatwould be considered an effective amount may be based on factorsindividual to each organ, including the type of organ, disease or injurybeing treated, the way the organ has been processed, length of time fromcollection, etc. One skilled in the art will be able to determine theeffective amount for a given organ based on these considerations whichare routine in the art. As used herein, “effective dose” means the sameas “effective amount.”

“Effective route” generally means a route which provides for delivery ofan agent to a desired compartment, system, or location. For example, aneffective route is one through which an agent can be administered toprovide at the desired site of action an amount of the agent sufficientto effectuate a beneficial or desired clinical result (in the presentcase, effective transplantation).

“Embryonic Stem Cells (ESC)” are well known in the art and have beenprepared from many different mammalian species. Embryonic stem cells arestem cells derived from the inner cell mass of an early stage embryoknown as a blastocyst. They are able to differentiate into allderivatives of the three primary germ layers: ectoderm, endoderm, andmesoderm. These include each of the more than 220 cell types in theadult body. The ES cells can become any tissue in the body, excludingplacenta. Only the morula's cells are totipotent, able to become alltissues and a placenta. Some cells similar to ESCs may be produced bynuclear transfer of a somatic cell nucleus into an enucleated fertilizedegg.

The term “exogenous”, when used in relation to a stem cell, generallyrefers to a stem cell that is external to the organ and which has beenexposed to (e.g., contacted with) an organ intended for transplantationby an effective route. An exogenous stem cell may be from the samesubject or from a different subject. In one embodiment, exogenous stemcells can include stem cells that have been harvested from a subject,isolated, expanded ex vivo, and then exposed to an organ intended fortransplantation by an effective route.

The term “expose” can include the act of administering one or more stemcells to an organ intended for transplantation. Administration to theorgan can be done ex vivo or in vivo (e.g., by perfusion into asubject).

Use of the term “includes” is not intended to be limiting.

“Increase” or “increasing” means to induce a biological event entirelyor to increase the degree of the event.

“Induced pluripotent stem cells (IPSC or IPS cells)” are somatic cellsthat have been reprogrammed, for example, by introducing exogenous genesthat confer on the somatic cell a less differentiated phenotype. Thesecells can then be induced to differentiate into less differentiatedprogeny. IPS cells have been derived using modifications of an approachoriginally discovered in 2006 (Yamanaka, S. et al., Cell Stem Cell,1:39-49 (2007)). For example, in one instance, to create IPS cells,scientists started with skin cells that were then modified by a standardlaboratory technique using retroviruses to insert genes into thecellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4,and c-myc, known to act together as natural regulators to keep cells inan embryonic stem cell-like state. These cells have been described inthe literature. See, for example, Wernig et al., PNAS, 105:5856-5861(2008); Jaenisch et al., Cell, 132:567-582 (2008); Hanna et al., Cell,133:250-264 (2008); and Brambrink et al., Cell Stem Cell, 2:151-159(2008). These references are incorporated by reference for teachingIPSCs and methods for producing them. It is also possible that suchcells can be created by specific culture conditions (exposure tospecific agents).

The term “ischemic reperfusion injury” is understood in the industry andis described for example inhttp://emedicine.medscape.com/article/431140-overview#aw2aab 6b3 (aboutOrgan Preservation), as well as de Groot, H. et al., Transplant Proc.39(2):481-4 (March 2007), which are incorporated herein by reference forthe teaching of ischemic reperfusion injury and its mechanistic details.

“Ischemia” occurs in two phases. The first phase is referred to as thewarm ischemic phase and includes the time from which the donor organ isremoved and circulation is interrupted to the time that the organ isadministered with a hypothermic preservation solution. The cold ischemicphase occurs when the organ is preserved in a hypothermic state prior totransplantation and normal recirculation in the recipient.

The term “isolated” refers to a cell or cells which are not associatedwith one or more cells or one or more cellular components that areassociated with the cell or cells in vivo. An “enriched population”means a relative increase in numbers of a desired cell relative to oneor more other cell types in vivo or in primary culture.

However, as used herein, the term “isolated” does not indicate thepresence of only the cells of the invention. Rather, the term “isolated”indicates that the cells of the invention are removed from their naturaltissue environment and are present at a higher concentration as comparedto the normal tissue environment. Accordingly, an “isolated” cellpopulation may further include cell types in addition to the cells ofthe invention cells and may include additional tissue components. Thisalso can be expressed in terms of cell doublings, for example. A cellmay have undergone 10, 20, 30, 40 or more doublings in vitro or ex vivoso that it is enriched compared to its original numbers in vivo or inits original tissue environment (e.g., bone marrow, peripheral blood,placenta, umbilical cord, umbilical cord blood, adipose tissue, etc.).

“MAPC” is an acronym for “multipotent adult progenitor cell.” It refersto a cell that is not an embryonic stem cell or germ cell but has somecharacteristics of these. MAPC can be characterized in a number ofalternative descriptions, each of which conferred novelty to the cellswhen they were discovered. They can, therefore, be characterized by oneor more of those descriptions. First, they have extended replicativecapacity in culture without being transformed (tumorigenic) and with anormal karyotype. Second, they may give rise to cell progeny of morethan one germ layer, such as two or all three germ layers (i.e.,endoderm, mesoderm and ectoderm) upon differentiation. Third, althoughthey are not embryonic stem cells or germ cells, they may expressmarkers of these primitive cell types so that MAPCs may express one ormore of Oct 3/4 (i.e., Oct 3A), rex-1, and rox-1. They may also expressone or more of sox-2 and SSEA-4. Fourth, like a stem cell, they mayself-renew, that is, have an extended replication capacity without beingtransformed. This means that these cells express telomerase (i.e., havetelomerase activity). Accordingly, the cell type that was designated“MAPC” may be characterized by alternative basic characteristics thatdescribe the cell via some of its novel properties.

The term “adult” in MAPC is non-restrictive. It refers to anon-embryonic somatic cell. MAPCs are karyotypically normal and do notform teratomas in vivo. This acronym was first used in U.S. Pat. No.7,015,037 to describe a pluripotent cell isolated from bone marrow.However, cells with pluripotential markers and/or differentiationpotential have been discovered subsequently and, for purposes of thisinvention, may be equivalent to those cells first designated “MAPC.”Essential descriptions of the MAPC type of cell are provided in theSummary of the Invention above.

MAPC represents a more primitive progenitor cell population than MSC(Verfaillie, C. M., Trends Cell Biol 12:502-8 (2002), Jahagirdar, B. N.,et al., Exp Hematol, 29:543-56 (2001); Reyes, M. and C. M. Verfaillie,Ann N Y Acad Sci, 938:231-233 (2001); Jiang, Y. et al., Exp Hematol,30896-904 (2002); and Jiang, Y. et al., Nature, 418:41-9. (2002)).

The term “MultiStem®” is the trade name for a cell preparation based onthe MAPCs of U.S. Pat. No. 7,015,037, i.e., a non-embryonic stem,non-germ cell as described above. MultiStem® is prepared according tocell culture methods disclosed in this patent application, particularly,lower oxygen and higher serum. MultiStem® is highly expandable,karyotypically normal, and does not form teratomas in vivo. It maydifferentiate into cell lineages of more than one germ layer and mayexpress one or more of telomerase, oct3/4, rex-1, rox-1, sox-2, andSSEA4.

The term “organ” may be used according to its customary and understoodmeaning in the industry as an entire intact organ that has been removedfrom the donor for transplantation or is intended to be removed from thedonor for transplantation into a recipient. Although the term “organ” isemphasized in this application, the methods apply to tissues that maynot constitute whole organs. That is, to parts of organs such as thosedisclosed elsewhere in this application. Therefore, where appropriate,the term “tissue” can be appropriately substituted for the term “organ”.

“Pharmaceutically-acceptable carrier” is any pharmaceutically-acceptablemedium for the cells used in the present invention. Such a medium mayretain isotonicity, cell metabolism, pH, and the like. It is compatiblewith administration to an organ and can be used, therefore, for celldelivery and treatment.

The term “potency” refers to the ability of the cells to achieve theeffects described in this application. Accordingly, potency refers tothe effect at various levels, including, but not limited to, increasingthe probability of a successful transplantation, retarding thedeterioration of a pre-transplant organ, reducing inflammatory activityin the organ, providing immunological tolerance to the organ, increasingthe production of anti-inflammatory cytokines in the organ, increasingthe presence of neuroprotective T-cells in the organ, decreasing thepresence of reactive T-cells in the organ, reducing the level ofpro-inflammatory cytokines in the organ, reducing the effects of hypoxiain the organ, reversing the level of edema in the organ, and reducingthe effects of hypothermia in the organ. Injury that is sustained duringrecovery, preservation, and transplantation, occurs mainly from ischemiaand hypothermia. These can affect the organs in various ways. These aredescribed in the Medscape reference cited above and the link is given inthis application. According to that reference, the mechanisms of tissueinjury include a loss of integrity in the cell structure, disruption ofthe ionic composition of the cell, disruption in ATP generation, and, asa result of reperfusion, damage may occur during reperfusion by thetoxic accumulation of oxygen free radicals.

With respect to integrity of the cell structure, integrity may beinterrupted by loss of structural integrity in the cell membrane.Maintaining the integrity of the cell membrane depends on control oftemperature, pH, osmolarity. Organ ischemia and preservation disrupt allof these parameters.

“Primordial embryonic germ cells” (PG or EG cells) can be cultured andstimulated to produce many less differentiated cell types.

“Progenitor cells” are cells produced during differentiation of a stemcell that have some, but not all, of the characteristics of theirterminally-differentiated progeny. Defined progenitor cells, such as“cardiac progenitor cells,” are committed to a lineage, but not to aspecific or terminally differentiated cell type. The term “progenitor”as used in the acronym “MAPC” does not limit these cells to a particularlineage. A progenitor cell can form a progeny cell that is more highlydifferentiated than the progenitor cell.

The term “reduce” as used herein means to prevent as well as decrease.In the context of organ treatment, to “reduce” is to either prevent orameliorate organ rejection. This includes causes or symptoms of organrejection. This applies, for example, to the underlying biological causeof rejection, such as, ameliorating the deleterious effects ofinflammation.

“Selecting” a cell with a desired level of potency can mean identifying(as by assay), isolating, and expanding a cell. This could create apopulation that has a higher potency than the parent cell populationfrom which the cell was isolated. The “parent” cell population refers tothe parent cells from which the selected cells divided. “Parent” refersto an actual P1→F1 relationship (i.e., a progeny cell). So if cell X isisolated from a mixed population of cells X and Y, in which X is anexpressor and Y is not, one would not classify a mere isolate of X ashaving enhanced expression. But, if a progeny cell of X is a higherexpressor, one would classify the progeny cell as having enhancedexpression.

To select a cell that achieves the desired effect would include both anassay to determine if the cells achieve the desired effect and wouldalso include obtaining those cells. The cell may naturally achieve thedesired effect in that the effect is not achieved by an exogenoustransgene/DNA. But an effective cell may be improved by being incubatedwith or exposed to an agent that increases the effect. The cellpopulation from which the effective cell is selected may not be known tohave the potency prior to conducting the assay. The cell may not beknown to achieve the desired effect prior to conducting the assay. As aneffect could depend on gene expression and/or secretion, one could alsoselect on the basis of one or more of the genes that cause the effect.

Selection could be from cells in a tissue. For example, in this case,cells would be isolated from a desired tissue, expanded in culture,selected for achieving the desired effect, and the selected cellsfurther expanded.

Selection could also be from cells ex vivo, such as cells in culture. Inthis case, one or more of the cells in culture would be assayed forachieving the desired effect and the cells obtained that achieve thedesired effect could be further expanded.

Cells could also be selected for enhanced ability to achieve the desiredeffect. In this case, the cell population from which the enhanced cellis obtained already has the desired effect. Enhanced effect means ahigher average amount per cell than in the parent population.

The parent population from which the enhanced cell is selected may besubstantially homogeneous (the same cell type). One way to obtain suchan enhanced cell from this population is to create single cells or cellpools and assay those cells or cell pools to obtain clones thatnaturally have the enhanced (greater) effect (as opposed to treating thecells with a modulator that induces or increases the effect) and thenexpanding those cells that are naturally enhanced.

However, cells may be treated with one or more agents that will induceor increase the effect. Thus, substantially homogeneous populations maybe treated to enhance the effect.

If the population is not substantially homogeneous, then, it ispreferable that the parental cell population to be treated contains atleast 100 of the desired cell type in which enhanced effect is sought,more preferably at least 1,000 of the cells, and still more preferably,at least 10,000 of the cells. Following treatment, this sub-populationcan be recovered from the heterogeneous population by known cellselection techniques and further expanded if desired.

Thus, desired levels of effect may be those that are higher than thelevels in a given preceding population. For example, cells that are putinto primary culture from a tissue and expanded and isolated by cultureconditions that are not specifically designed to produce the effect mayprovide a parent population. Such a parent population can be treated toenhance the average effect per cell or screened for a cell or cellswithin the population that express greater degrees of effect withoutdeliberate treatment. Such cells can be expanded then to provide apopulation with a higher (desired) expression.

“Self-renewal” of a stem cell refers to the ability to produce replicatedaughter stem cells having differentiation potential that is identicalto those from which they arose. A similar term used in this context is“proliferation.”

“Stem cell” means a cell that can undergo self-renewal (i.e., progenywith the same differentiation potential) and also produce progeny cellsthat are more restricted in differentiation potential. Within thecontext of the invention, a stem cell would also encompass a moredifferentiated cell that has de-differentiated, for example, by nucleartransfer, by fusion with a more primitive stem cell, by introduction ofspecific transcription factors, or by culture under specific conditions.See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying etal., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1199-1203(2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al.,Nature, 448:313-317 (2007); and Takahashi et al., Cell, 131:861-872(2007).

Dedifferentiation may also be caused by the administration of certaincompounds or exposure to a physical environment in vitro or in vivo thatwould cause the dedifferentiation. Stem cells also may be derived fromabnormal tissue, such as a teratocarcinoma and some other sources suchas embryoid bodies (although these can be considered embryonic stemcells in that they are derived from embryonic tissue, although notdirectly from the inner cell mass). Stem cells may also be produced byintroducing genes associated with stem cell function into a non-stemcell, such as an induced pluripotent stem cell.

“Subject” means a vertebrate, such as a mammal, such as a human. Mammalsinclude, but are not limited to, humans, dogs, cats, horses, cows, andpigs.

The term “therapeutically effective amount” refers to the amount of anagent determined to produce any therapeutic response in a mammal. Forexample, effective anti-inflammatory therapeutic agents may prolong thesurvivability of the patient, and/or inhibit overt clinical symptoms.Treatments that are therapeutically effective within the meaning of theterm as used herein, include treatments that improve a subject's qualityof life even if they do not improve the disease outcome per se. Suchtherapeutically effective amounts are readily ascertained by one ofordinary skill in the art. Thus, to “treat” means to deliver such anamount. Thus, treating can prevent or ameliorate any pathologicalsymptoms.

The term “tolerization” or “tolerize” refers to the treatment of thepre-transplantation organ (graft) with the stem cells to reduce theimmunogenicity of the graft to enable or facilitate the development oftolerance of the organ by the recipient. The term broadly refers to theconcept of reducing the immunogenicity of the transplant organ, whichenables or facilitates tolerance development by the recipient. Thus,tolerizing the organ causes the organ to be tolerated by the recipient.In other words, the term can refer to making the immune system unable toelicit an immune response to a cell or tissue that normally elicits animmune response. An example of this is when a T-regulatory cell secretesfactors that suppress an activated T-cell so that I can no longersecreted pro-inflammatory cytokines.

This might be accomplished via ex vivo treatment prior totransplantation, or even local administration prior to harvest.

“Treat,” “treating,” or “treatment” are used broadly in relation to theinvention and each such term encompasses, among others, preventing,ameliorating, inhibiting, or curing a deficiency, dysfunction, disease,or other deleterious process, including those that interfere with and/orresult from a therapy.

“Validate” means to confirm. In the context of the invention, oneconfirms that a cell is an expressor with a desired potency. This is sothat one can then use that cell (in treatment, banking, drug screening,etc.) with a reasonable expectation of efficacy. Accordingly, tovalidate means to confirm that the cells, having been originally foundto have/established as having the desired activity, in fact, retain thatactivity. Thus, validation is a verification event in a two-eventprocess involving the original determination and the follow-updetermination. The second event is referred to herein as “validation.”

Stem Cells

The present invention can be practiced, preferably, using stem cells ofvertebrate species, such as humans, non-human primates, domesticanimals, livestock, and other non-human mammals. These include, but arenot limited to, those cells described below.

Embryonic Stem Cells

The most well studied stem cell is the embryonic stem cell (ESC) as ithas unlimited self-renewal and multipotent differentiation potential.These cells are derived from the inner cell mass of the blastocyst orcan be derived from the primordial germ cells of a post-implantationembryo (embryonal germ cells or EG cells). ES and EG cells have beenderived, first from mouse, and later, from many different animals, andmore recently, also from non-human primates and humans. When introducedinto mouse blastocysts or blastocysts of other animals, ESCs cancontribute to all tissues of the animal ES and EG cells can beidentified by positive staining with antibodies against SSEA1 (mouse)and SSEA4 (human) See, for example, U.S. Pat. Nos. 5,453,357; 5,656,479;5,670,372; 5,843,780; 5,874,301; 5,914,268; 6,110,739 6,190,910;6,200,806; 6,432,711; 6,436,701, 6,500,668; 6,703,279; 6,875,607;7,029,913; 7,112,437; 7,145,057; 7,153,684; and 7,294,508, each of whichis incorporated by reference for teaching embryonic stem cells andmethods of making and expanding them. Accordingly, ESCs and methods forisolating and expanding them are well-known in the art.

A number of transcription factors and exogenous cytokines have beenidentified that influence the potency status of embryonic stem cells invivo. The first transcription factor to be described that is involved instem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit-Oct-Unc)family of transcription factors and is a DNA binding protein that isable to activate the transcription of genes, containing an octamericsequence called “the octamer motif” within the promoter or enhancerregion. Oct4 is expressed at the moment of the cleavage stage of thefertilized zygote until the egg cylinder is formed. The function ofOct3/4 is to repress differentiation inducing genes (i.e., FoxaD3, hCG)and to activate genes promoting pluripotency (FGF4, Utf1, Rexl). Sox2, amember of the high mobility group (HMG) box transcription factors,cooperates with Oct4 to activate transcription of genes expressed in theinner cell mass. It is essential that Oct3/4 expression in embryonicstem cells is maintained between certain levels. Overexpression ordownregulation of >50% of Oct4 expression level will alter embryonicstem cell fate, with the formation of primitive endoderm/mesoderm ortrophectoderm, respectively. In vivo, Oct4 deficient embryos develop tothe blastocyst stage, but the inner cell mass cells are not pluripotent.Instead they differentiate along the extraembryonic trophoblast lineage.Sall4, a mammalian Spalt transcription factor, is an upstream regulatorof Oct4, and is therefore important to maintain appropriate levels ofOct4 during early phases of embryology. When Sall4 levels fall below acertain threshold, trophectodermal cells will expand ectopically intothe inner cell mass. Another transcription factor required forpluripotency is Nanog, named after a celtic tribe “Tir Nan Og”: the landof the ever young. In vivo, Nanog is expressed from the stage of thecompacted morula, is subsequently defined to the inner cell mass and isdownregulated by the implantation stage. Downregulation of Nanog may beimportant to avoid an uncontrolled expansion of pluripotent cells and toallow multilineage differentiation during gastrulation. Nanog nullembryos, isolated at day 5.5, consist of a disorganized blastocyst,mainly containing extraembryonic endoderm and no discernible epiblast.

Non-Embryonic Stem Cells

Stem cells have been identified in most tissues. Perhaps the bestcharacterized is the hematopoietic stem cell (HSC). HSCs aremesoderm-derived cells that can be purified using cell surface markersand functional characteristics. They have been isolated from bonemarrow, peripheral blood, cord blood, fetal liver, and yolk sac. Theyinitiate hematopoiesis and generate multiple hematopoietic lineages.When transplanted into lethally-irradiated animals, they can repopulatethe erythroid neutrophil-macrophage, megakaryocyte, and lymphoidhematopoietic cell pool. They can also be induced to undergo someself-renewal cell division. See, for example, U.S. Pat. Nos. 5,635,387;5,460,964; 5,677,136; 5,750,397; 5,681,599; and 5,716,827. U.S. Pat. No.5,192,553 reports methods for isolating human neonatal or fetalhematopoietic stem or progenitor cells. U.S. Pat. No. 5,716,827 reportshuman hematopoietic cells that are Thy-1+ progenitors, and appropriategrowth media to regenerate them in vitro. U.S. Pat. No. 5,635,387reports a method and device for culturing human hematopoietic cells andtheir precursors. U.S. Pat. No. 6,015,554 describes a method ofreconstituting human lymphoid and dendritic cells. Accordingly, HSCs andmethods for isolating and expanding them are well-known in the art.

Another stem cell that is well-known in the art is the neural stem cell(NSC). These cells can proliferate in vivo and continuously regenerateat least some neuronal cells. When cultured ex vivo, neural stem cellscan be induced to proliferate as well as differentiate into differenttypes of neurons and glial cells. When transplanted into the brain,neural stem cells can engraft and generate neural and glial cells. See,for example, Gage F. H., Science, 287:1433-1438 (2000), Svendsen S. N.et al., Brain Pathology, 9:499-513 (1999), and Okabe S. et al., MechDevelopment, 59:89-102 (1996). U.S. Pat. No. 5,851,832 reportsmultipotent neural stem cells obtained from brain tissue. U.S. Pat. No.5,766,948 reports producing neuroblasts from newborn cerebralhemispheres. U.S. Pat. Nos. 5,564,183 and 5,849,553 report the use ofmammalian neural crest stem cells. U.S. Pat. No. 6,040,180 reports invitro generation of differentiated neurons from cultures of mammalianmultipotential CNS stem cells. WO 98/50526 and WO 99/01159 reportgeneration and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain. Accordingly, neural stem cells and methods formaking and expanding them are well-known in the art.

Another stem cell that has been studied extensively in the art is themesenchymal stem cell (MSC). MSCs are derived from the embryonalmesoderm and can be isolated from many sources, including adult bonemarrow, peripheral blood, fat, placenta, and umbilical blood, amongothers. MSCs can differentiate into many mesodermal tissues, includingmuscle, bone, cartilage, fat, and tendon. There is considerableliterature on these cells. See, for example, U.S. Pat. Nos. 5,486,389;5,827,735; 5,811,094; 5,736,396; 5,837,539; 5,837,670; and 5,827,740.See also Pittenger, M. et al., Science, 284:143-147 (1999).

Another example of an adult stem cell is adipose-derived adult stemcells (ADSCs) which have been isolated from fat, typically byliposuction followed by release of the ADSCs using collagenase. ADSCsare similar in many ways to MSCs derived from bone marrow, except thatit is possible to isolate many more cells from fat. These cells havebeen reported to differentiate into bone, fat, muscle, cartilage, andneurons. A method of isolation has been described in U.S. PatentPublication No. 2005/0153442 A1.

Other stem cells that are known in the art include gastrointestinal stemcells, epidermal stem cells, and hepatic stem cells, which have alsobeen termed “oval cells” (Potten, C., et al., Trans R Soc Lond B BiolSci, 353:821-830 (1998), Watt, F., Trans R Soc Lond B Biol Sci, 353:831(1997); Alison et al., Hepatology, 29:678-683 (1998).

Other non-embryonic cells reported to be capable of differentiating intocell types of more than one embryonic germ layer include, but are notlimited to, cells from umbilical cord blood (see U.S. Publication No.2002/0164794), placenta (see U.S. Publication No. 2003/0181269,umbilical cord matrix (Mitchell, K. E. et al., Stem Cells, 21:50-60(2003)), small embryonic-like stem cells (Kucia, M. et al., J PhysiolPharmacol, 57 Suppl 5:5-18 (2006)), amniotic fluid stem cells (Atala,A., J Tissue Regen Med, 1:83-96 (2007)), skin-derived precursors (Tomaet al., Nat Cell Biol, 3:778-784 (2001)), bone marrow (see U.S. PatentPublication Nos. 2003/0059414 and 2006/0147246), marrow-isolated adultmultilineage inducible (MIAMI) cells (see PCT/US2004/002580), andendometrial cells (see U.S. Publication No. 2013/0156726), each of whichis incorporated by reference for teaching these cells.

Strategies of Reprogramming Somatic Cells

Several different strategies, such as nuclear transplantation, cellularfusion, and culture induced reprogramming have been employed to inducethe conversion of differentiated cells into an embryonic state. Nucleartransfer involves the injection of a somatic nucleus into an enucleatedoocyte, which, upon transfer into a surrogate mother, can give rise to aclone (“reproductive cloning”), or, upon explantation in culture, cangive rise to genetically matched embryonic stem (ES) cells (“somaticcell nuclear transfer,” SCNT). Cell fusion of somatic cells with EScells results in the generation of hybrids that show all features ofpluripotent ES cells. Explantation of somatic cells in culture selectsfor immortal cell lines that may be pluripotent or multipotent. Atpresent, spermatogonial stem cells are the only source of pluripotentcells that can be derived from postnatal animals. Transduction ofsomatic cells with defined factors can initiate reprogramming to apluripotent state. These experimental approaches have been extensivelyreviewed (Hochedlinger and Jaenisch, Nature, 441:1061-1067 (2006) andYamanaka, S., Cell Stem Cell, 1:39-49 (2007)).

Nuclear Transfer

Nuclear transplantation (NT), also referred to as somatic cell nucleartransfer (SCNT), denotes the introduction of a nucleus from a donorsomatic cell into an enucleated ogocyte to generate a cloned animal suchas Dolly the sheep (Wilmut et al., Nature, 385:810-813 (1997). Thegeneration of live animals by NT demonstrated that the epigenetic stateof somatic cells, including that of terminally differentiated cells,while stable, is not irreversible fixed but can be reprogrammed to anembryonic state that is capable of directing development of a neworganism. In addition to providing an exciting experimental approach forelucidating the basic epigenetic mechanisms involved in embryonicdevelopment and disease, nuclear cloning technology is of potentialinterest for patient-specific transplantation medicine.

Fusion of Somatic Cells and Embryonic Stem Cells

Epigenetic reprogramming of somatic nuclei to an undifferentiated statehas been demonstrated in murine hybrids produced by fusion of embryoniccells with somatic cells. Hybrids between various somatic cells andembryonic carcinoma cells (Solter, D., Nat Rev Genet, 7:319-327 (2006),embryonic germ (EG), or ES cells (Zwaka and Thomson, Development,132:227-233 (2005)) share many features with the parental embryoniccells, indicating that the pluripotent phenotype is dominant in suchfusion products. As with mouse (Tada et al., Curr Biol, 11:1553-1558(2001)), human ES cells have the potential to reprogram somatic nucleiafter fusion (Cowan et al., Science, 309:1369-1373(2005)); Yu et al.,Science, 318:1917-1920 (2006)). Activation of silent pluripotencymarkers such as Oct4 or reactivation of the inactive somatic Xchromosome provided molecular evidence for reprogramming of the somaticgenome in the hybrid cells. It has been suggested that DNA replicationis essential for the activation of pluripotency markers, which is firstobserved 2 days after fusion (Do and Scholer, Stem Cells, 22:941-949(2004)), and that forced overexpression of Nanog in ES cells promotespluripotency when fused with neural stem cells (Silva et al., Nature,441:997-1001 (2006)).

Culture-Induced Reprogramming

Pluripotent cells have been derived from embryonic sources such asblastomeres and the inner cell mass (ICM) of the blastocyst (ES cells),the epiblast (EpiSC cells), primordial germ cells (EG cells), andpostnatal spermatogonial stem cells (“maGSCsm” “ES-like” cells). Thefollowing pluripotent cells, along with their donor cell/tissue is asfollows: parthogenetic ES cells are derived from murine oocytes(Narasimha et al., Curr Biol, 7:881-884 (1997)); embryonic stem cellshave been derived from blastomeres (Wakayama et al., Stem Cells,25:986-993 (2007)); inner cell mass cells (source not applicable) (Egganet al., Nature, 428:44-49 (2004)); embryonic germ and embryonalcarcinoma cells have been derived from primordial germ cells (Matsui etal., Cell, 70:841-847 (1992)); GMCS, maSSC, and MASC have been derivedfrom spermatogonial stem cells (Guan et al., Nature, 440:1199-1203(2006); Kanatsu-Shinohara et al., Cell, 119:1001-1012 (2004); andSeandel et al., Nature, 449:346-350 (2007)); EpiSC cells are derivedfrom epiblasts (Brons et al., Nature, 448:191-195 (2007); Tesar et al.,Nature, 448:196-199(2007)); parthogenetic ES cells have been derivedfrom human oocytes (Cibelli et al., Science, 295L819 (2002); Revazova etal., Cloning Stem Cells, 9:432-449 (2007)); human ES cells have beenderived from human blastocysts (Thomson et al., Science, 282:1145-1147(1998)); MAPC have been derived from bone marrow (Jiang et al., Nature,418:41-49 (2002); Phinney and Prockop, Stem Cells, 25:2896-2902 (2007));cord blood cells (derived from cord blood) (van de Ven et al., ExpHematol, 35:1753-1765 (2007)); neurosphere derived cells derived fromneural cell (Clarke et al., Science, 288:1660-1663 (2000)). Donor cellsfrom the germ cell lineage such as PGCs or spermatogonial stem cells areknown to be unipotent in vivo, but it has been shown that pluripotentES-like cells (Kanatsu-Shinohara et al., Cell, 119:1001-1012 (2004) ormaGSCs (Guan et al., Nature, 440:1199-1203 (2006), can be isolated afterprolonged in vitro culture. While most of these pluripotent cell typeswere capable of in vitro differentiation and teratoma formation, onlyES, EG, EC, and the spermatogonial stem cell-derived maGCSs or ES-likecells were pluripotent by more stringent criteria, as they were able toform postnatal chimeras and contribute to the germline Recently,multipotent adult spermatogonial stem cells (MASCs) were derived fromtesticular spermatogonial stem cells of adult mice, and these cells hadan expression profile different from that of ES cells (Seandel et al.,Nature, 449:346-350 (2007)) but similar to EpiSC cells, which werederived from the epiblast of postimplantation mouse embryos (Brons etal., Nature, 448:191-195 (2007); Tesar et al., Nature, 448:196-199(2007)).

Reprogramming by Defined Transcription Factors

Takahashi and Yamanaka have reported reprogramming somatic cells back toan ES-like state (Takahashi and Yamanaka, Cell, 126:663-676 (2006)).They successfully reprogrammed mouse embryonic fibroblasts (MEFs) andadult fibroblasts to pluripotent ES-like cells after viral-mediatedtransduction of the four transcription factors Oct4, Sox2, c-myc, andKlf4 followed by selection for activation of the Oct4 target gene Fbx15.Cells that had activated Fbx15 were coined iPS (induced pluripotentstem) cells and were shown to be pluripotent by their ability to formteratomas, although they were unable to generate live chimeras. Thispluripotent state was dependent on the continuous viral expression ofthe transduced Oct4 and Sox2 genes, whereas the endogenous Oct4 andNanog genes were either not expressed or were expressed at a lower levelthan in ES cells, and their respective promoters were found to belargely methylated. This is consistent with the conclusion that theFbx15-iPS cells did not correspond to ES cells but may have representedan incomplete state of reprogramming. While genetic experiments hadestablished that Oct4 and Sox2 are essential for pluripotency (Chambersand Smith, Oncogene, 23:7150-7160 (2004); Ivanona et al., Nature,442:5330538 (2006); Masui et al., Nat Cell Biol, 9:625-635 (2007)), therole of the two oncogenes c-myc and Klf4 in reprogramming is less clear.Some of these oncogenes may, in fact, be dispensable for reprogramming,as both mouse and human iPS cells have been obtained in the absence ofc-myc transduction, although with low efficacy (Nakagawa et al., NatBiotechnol, 26:191-106 (2008); Werning et al., Nature, 448:318-324(2008); Yu et al., Science, 318: 1917-1920 (2007)).

MAPC

Human MAPCs are described in U.S. Pat. No. 7,015,037. MAPCs have beenidentified in other mammals. Murine MAPCs, for example, are alsodescribed in U.S. Pat. No. 7,015,037. Rat MAPCs are also described inU.S. Pat. No. 7,838,289.

These references are incorporated by reference for describing MAPCsfirst isolated by Catherine Verfaillie.

Isolation and Growth of MAPCs

Methods of MAPC isolation are known in the art. See, for example, U.S.Pat. No. 7,015,037, and these methods, along with the characterization(phenotype) of MAPCs, are incorporated herein by reference. MAPCs can beisolated from multiple sources, including, but not limited to, bonemarrow, placenta, umbilical cord and cord blood, muscle, brain, liver,spinal cord, blood or skin. It is, therefore, possible to obtain bonemarrow aspirates, brain or liver biopsies, and other organs, and isolatethe cells using positive or negative selection techniques available tothose of skill in the art, relying upon the genes that are expressed (ornot expressed) in these cells (e.g., by functional or morphologicalassays such as those disclosed in the above-referenced applications,which have been incorporated herein by reference).

MAPCs have also been obtained my modified methods described in Breyer etal., Experimental Hematology, 34:1596-1601 (2006) and Subramanian etal., Cellular Programming and Reprogramming: Methods and Protocols; S.Ding (ed.), Methods in Molecular Biology, 636:55-78 (2010), incorporatedby reference for these methods.

MAPCs from Human Bone Marrow as Described in U.S. Pat. No. 7,015,037

MAPCs do not express the common leukocyte antigen CD45 or erythroblastspecific glycophorin-A (Gly-A). The mixed population of cells wassubjected to a Ficoll Hypaque separation. The cells were then subjectedto negative selection using anti-CD45 and anti-Gly-A antibodies,depleting the population of CD45+ and Gly-A+ cells, and the remainingapproximately 0.1% of marrow mononuclear cells were then recovered.Cells could also be plated in fibronectin-coated wells and cultured asdescribed below for 2-4 weeks to deplete the cells of CD45+ and Gly-A+cells. In cultures of adherent bone marrow cells, many adherent stromalcells undergo replicative senescence around cell doubling 30 and a morehomogenous population of cells continues to expand and maintains longtelomeres.

Alternatively, positive selection could be used to isolate cells via acombination of cell-specific markers. Both positive and negativeselection techniques are available to those of skill in the art, andnumerous monoclonal and polyclonal antibodies suitable for negativeselection purposes are also available in the art (see, for example,Leukocyte Typing V, Schlossman, et al., Eds. (1995) Oxford UniversityPress) and are commercially available from a number of sources.

Techniques for mammalian cell separation from a mixture of cellpopulations have also been described by Schwartz, et al., in U.S. Pat.No. 5,759,793 (magnetic separation), Basch et al., 1983 (immunoaffinitychromatography), and Wysocki and Sato, 1978 (fluorescence-activated cellsorting).

Cells may be cultured in low-serum or serum-free culture medium.Serum-free medium used to culture MAPCs is described in U.S. Pat. No.7,015,037. Commonly-used growth factors include but are not limited toplatelet-derived growth factor and epidermal growth factor. See, forexample, U.S. Pat. Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161;6,617,159; 6,372,210; 6,224,860; 6,037,174; 5,908,782; 5,766,951;5,397,706; and 4,657,866; all incorporated by reference for teachinggrowing cells in serum-free medium.

Additional Culture Methods

In additional experiments, the density at which MAPCs are cultured canvary from about 100 cells/cm² or about 150 cells/cm² to about 10,000cells/cm², including about 200 cells/cm² to about 1500 cells/cm² toabout 2000 cells/cm². The density can vary between species.Additionally, optimal density can vary depending on culture conditionsand source of cells. It is within the skill of the ordinary artisan todetermine the optimal density for a given set of culture conditions andcells.

Also, effective atmospheric oxygen concentrations of less than about10%, including about 1-5% and, especially, 3-5%, can be used at any timeduring the isolation, growth and differentiation of MAPCs in culture.

Cells may be cultured under various serum concentrations, e.g., about2-20%. Fetal bovine serum may be used. Higher serum may be used incombination with lower oxygen tensions, for example, about 15-20%. Cellsneed not be selected prior to adherence to culture dishes. For example,after a Ficoll gradient, cells can be directly plated, e.g.,250,000-500,000/cm². Adherent colonies can be picked, possibly pooled,and expanded.

In one embodiment, used in the experimental procedures in the Examples,high serum (around 15-20%) and low oxygen (around 3-5%) conditions wereused for the cell culture. Specifically, adherent cells from colonieswere plated and passaged at densities of about 1700-2300 cells/cm² in18% serum and 3% oxygen (with PDGF and EGF).

In an embodiment specific for MAPCs, supplements are cellular factors orcomponents that allow MAPCs to retain the ability to differentiate intocell types of more than one embryonic lineage, such as all threelineages. This may be indicated by the expression of specific markers ofthe undifferentiated state, such as Oct 3/4 (Oct 3A) and/or markers ofhigh expansion capacity, such as telomerase.

Cell Culture

For all the components listed below, see U.S. Pat. No. 7,015,037, whichis incorporated by reference for teaching these components.

In general, cells useful for the invention can be maintained andexpanded in culture medium that is available and well-known in the art.Also contemplated is supplementation of cell culture medium withmammalian sera. Additional supplements can also be used advantageouslyto supply the cells with the necessary trace elements for optimal growthand expansion. Hormones can also be advantageously used in cell culture.Lipids and lipid carriers can also be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Also contemplated is the use of feeder cell layers.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components. Stem cells oftenrequire additional factors that encourage their attachment to a solidsupport, such as type I and type II collagen, chondroitin sulfate,fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin,poly-D and poly-L-lysine, thrombospondin and vitronectin. One embodimentof the present invention utilizes fibronectin. See, for example, Ohashiet al., Nature Medicine, 13:880-885 (2007); Matsumoto et al., JBioscience and Bioengineering, 105:350-354 (2008); Kirouac et al., CellStem Cell, 3:369-381 (2008); Chua et al., Biomaterials, 26:2537-2547(2005); Drobinskaya et al., Stem Cells, 26:2245-2256 (2008);Dvir-Ginzberg et al., FASEB J, 22:1440-1449 (2008); Turner et al., JBiomed Mater Res Part B: Appl Biomater, 82B:156-168 (2007); and Miyazawaet al., Journal of Gastroenterology and Hepatology, 22:1959-1964(2007)).

Cells may also be grown in “3D” (aggregated) cultures. An example isPCT/US2009/31528, filed Jan. 21, 2009.

Once established in culture, cells can be used fresh or frozen andstored as frozen stocks, using, for example, DMEM with 40% FCS and 10%DMSO. Other methods for preparing frozen stocks for cultured cells arealso available to those of skill in the art.

Pharmaceutical Formulations

U.S. Pat. No. 7,015,037 is incorporated by reference for teachingpharmaceutical formulations. In certain embodiments, the cellpopulations are present within a composition adapted for and suitablefor delivery, i.e., physiologically compatible.

Formulations would be oriented to degree of desired effect, such asreduction of inflammation, reduction of apoptosis, edema, etc.,upregulation of certain factors, etc.

In some embodiments the purity of the cells for administration to anorgan is about 100% (substantially homogeneous). In other embodiments itis 95% to 100%. In some embodiments it is 85% to 95%. Particularly, inthe case of admixtures with other cells, the percentage can be about10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%,60%-70%, 70%-80%, 80%-90%, or 90%-95%. Or isolation/purity can beexpressed in terms of cell doublings where the cells have undergone, forexample, 10-20, 20-30, 30-40, 40-50 or more cell doublings.

The choice of formulation for administering the cells will depend on avariety of factors. Prominent among these will be the species ofdonor/recipient, the nature of the organ being treated, the nature ofother therapies and agents that are being administered, the optimumroute for administration, survivability via the effective route, thedosing regimen, and other factors that will be apparent to those skilledin the art. For instance, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form.

Final formulations of the aqueous suspension of cells/medium willtypically involve adjusting the ionic strength of the suspension toisotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e.,about pH 6.8 to 7.5). The final formulation will also typically containa fluid lubricant.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions to beadministered in methods of the invention. Typically, any additives (inaddition to the cells) are present in an amount of 0.001 to 50 wt % insolution, such as in phosphate buffered saline. The active ingredient ispresent in the order of micrograms to milligrams, such as about 0.0001to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt %, and most preferably about0.05 to about 5 wt %.

In some embodiments cells are encapsulated for administration,particularly where encapsulation enhances the effectiveness or providesadvantages in handling and/or shelf life. Cells may be encapsulated bymembranes, as well as capsules. It is contemplated that any of the manymethods of cell encapsulation available may be employed.

A wide variety of materials may be used in various embodiments formicroencapsulation of cells. Such materials include, for example,polymer capsules, alginate-poly-L-lysine-alginate microcapsules, bariumpoly-L-lysine alginate capsules, barium alginate capsules,polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, andpolyethersulfone (PES) hollow fibers.

Techniques for microencapsulation of cells that may be used foradministration of cells are known to those of skill in the art and aredescribed, for example, in Chang, P., et al., 1999; Matthew, H. W., etal., 1991; Yanagi, K., et al., 1989; Cai Z. H., et al., 1988; Chang, T.M., 1992 and in U.S. Pat. No. 5,639,275 (which, for example, describes abiocompatible capsule for long-term maintenance of cells that stablyexpress biologically active molecules). Additional methods ofencapsulation are in European Patent Publication No. 301,777 and U.S.Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350;5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing areincorporated herein by reference in parts pertinent to encapsulation ofcells.

Certain embodiments incorporate cells into a polymer, such as abiopolymer or synthetic polymer. Examples of biopolymers include, butare not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen,and proteoglycans. Other factors, such as the cytokines discussed above,can also be incorporated into the polymer. In other embodiments of theinvention, cells may be incorporated in the interstices of athree-dimensional gel. A large polymer or gel, typically, will besurgically implanted. A polymer or gel that can be formulated in smallenough particles or fibers can be administered by other common, moreconvenient, non-surgical routes.

The dosage of the cells will vary within wide limits and will be fittedto the individual requirements in each particular case. The number ofcells will vary depending on the type, weight, and condition of theorgan, the number or frequency of administrations, and other variablesknown to those of skill in the art. The cells can be administered by aroute that is suitable for the tissue or organ. Examples of suitabledelivery routes can include intra-tracheal delivery (e.g., for lung),intravenous delivery, intra-arterial delivery (e.g., intra-coronary),direct injection into the organ, and intra-lymphatic system delivery.

The cells can be suspended in an appropriate excipient in aconcentration from about 0.01 to 1×10⁵ cells/ml, about 1×10⁵ cells/ml to10×10⁶ cells/ml, or about 10×10⁶ cells/ml to 5×10⁷ cells/ml. Suitableexcipients are those that are biologically and physiologicallycompatible with the cells and with the recipient organ, such as bufferedsaline solution or other suitable excipients. The composition foradministration can be formulated, produced, and stored according tostandard methods complying with proper sterility and stability.

Dosing

Doses (i.e., the number of cells) for humans or other mammals can bedetermined without undue experimentation by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart. The optimal dose to be used in accordance with various embodimentsof the invention will depend on numerous factors, including thefollowing: the disease being treated and its stage; the species of thedonor, their health, gender, age, weight, and metabolic rate; thedonor's immunocompetence; other therapies being administered; andexpected potential complications from the donor's history or genotype.The parameters may also include: whether the cells are syngeneic,autologous, allogeneic, or xenogeneic; their potency; the site and/ordistribution that must be targeted; and such characteristics of the sitesuch as accessibility to cells. Additional parameters includeco-administration with other factors (such as growth factors andcytokines). The optimal dose in a given situation also will take intoconsideration the way in which the cells are formulated, the way theyare administered (e.g., perfusion, intra-organ, etc.), and the degree towhich the cells will be localized at the target sites followingadministration.

Ultimately, the dose levels, timing, and frequency would be determinedby effectiveness. This will be measured by organ health and viability,and possibly by organ function and clinical measures post-transplant.Such measures will vary by organ. In one embodiment, they could includeorgan function measures. One could access measures of organ viabilityfor transplant via the clinical literature. In another embodiment, thelevel(s) or pattern(s) of certain markers (e.g., tissue mRNA levels,cytokine levels, and inflammatory cell numbers) can be assayed (e.g.,using qPCR) to determine effectiveness. For example, the level of IL-10mRNA from pulmonary tissue can be assayed by qPCR to determineeffectiveness. In another example, one might evaluate dose in lung byimpact on: cytokine levels; other inflammatory markers in fluids (e.g.,obtained by bronchoalveolar lavage); edema levels; hemodynamic andventilator measures; and evaluation of gas exchange in ex vivo(re)perfused lungs.

In various embodiments, cells/medium may be administered in an initialdose, and thereafter maintained by further administration. Cells may beadministered by one method initially, and thereafter administered by thesame method or one or more different methods. The levels can bemaintained by the ongoing administration of the cells/medium. Variousembodiments administer the cells either initially or to maintain theirlevel in the subject or both by intravenous injection. In a variety ofembodiments, other forms of administration are used, dependent upon thetype and condition of the organ and other factors, discussed elsewhereherein.

Cells/medium may be administered in many frequencies over a wide rangeof times. Generally lengths of treatment will be proportional to thelength of the collection and handling process, the effectiveness of thetherapies being applied, and the condition and response of the organbeing treated.

In other embodiments, cells can be administered (e.g., by intravenous,intra-arterial, intra-tracheal, direct injection, etc.) to a donorsubject prior to organ harvest. Depending upon the route ofadministration, the cells can be administered for a suitable period oftime (e.g., minutes to about 1-4 hours, about 4-8 hours, about 8-12hours, about 12-16 hours, about 16-20 hours, about 20-24 hours). Theorgan can be harvested after the period of time by conventional surgicaltechnique(s). After harvest, cells are contacted (e g., immediatelycontacted) with the organ for a time sufficient to allow the cells todistribute throughout the organ (e.g., less than about 1 hour, about 1-2hours, about 2-3 hours, about 3-4 hours). Cells can be contacted withthe organ by infusion and/or immersion of the organ into a bathcontaining the cells. Next, the organ can be stored on ice and/orattached to a reperfusion system, whereafter the organ is delivered tothe transplantation site.

Upon arrival at the transplantation site, the organ can be placed on areperfusion system (if it has not been done so already) containing thecells. The organ can then be infused with the cells for a period of time(e.g., less than about an hour, about 1-2 hours, about 2-3 hours, about3-4 hours) depending upon the organ and the reperfusion system. Duringtransplantation, cells can be contacted with the organ by infusion intothe recipient (e.g., intravenously), direct injection into the organ,and/or direct application to a surface of the organ. Infusion can bedone, for example, prior to closure of the vessel(s) entering andexiting the organ. During liver transplantation, for instance, cells maybe infused into the hepatic portal vein prior to attachment of the veinto the transplanted liver. Additionally or alternatively, cells can bedelivered to the organ after transplantation (e.g., after closure of thevessel(s) entering and exiting the organ) for a suitable period of time(e.g., minutes to about 1-4 hours, about 4-8 hours, about 8-12 hours,about 12-16 hours, about 16-20 hours, about 20-24 hours) depending uponthe delivery system and the organ.

Compositions

The invention is also directed to cell populations with specificpotencies for achieving any of the effects described herein. Asdescribed above, these populations are established by selecting forcells that have desired potency. These populations are used to makeother compositions, for example, a cell bank comprising populations withspecific desired potencies and pharmaceutical compositions containing acell population with a specific desired potency.

EXAMPLE

Methods

Lung Harvest and Ex Vivo Perfusion

Following research consent obtained by the local Organ ProcurementAgency, LifeGift, the discarded donated lungs were procured for thisstudy under an established IRB protocol at the Houston Methodist(IRB(2)1111-0205). Lungs from each of the five patients were procured ina standard fashion with antegrade Perfadex (Vitrolife AB, Gothenburg,Sweden) 60 ml/Kg flush plus retrograde Perfadex perfusion through thepulmonary veins. The lungs were then stored in plastic bags containing 1liter of Perfadex and were kept on ice during transport. Once the lungsarrived at the Houston Methodist, they were then stored in arefrigerator @ 4° C. for a total 8 hours of cold static storage in orderto induce cold ischemic injury.

Ex Vivo Lung Perfusion (EVLP) was performed with the CE-marked VivolineLS1 (Vivoline Medical AB, Lund, Sweden) (FIG. 1) (Wierup, P. et al., AnnThorac Surg 2006; 81(2):460-6; Ingemansson, R. et al., Ann Thorac Surg2009; 87(1):255-60; and Cypel, M. et al., N Engl J Med 2011;364(15):1431-40). The system was primed with 2.5 L of Steen Solution(XVIVO Perfusion). The use of washed red blood cell or blood was avoidedin order to decrease the number of variables in the feasibility study.Meropenem 100 mg (AstraZeneca AB, Sodertalje, Sweden) and 10,000 U ofHeparin (LEO Pharmaceutical, Copenhagen, Denmark) were added to theperfusate. Before the lungs were connected to the EVLP unit, the pH inthe solution was corrected to between 7.35 and 7.45 using trometamol(Addex-THAM, Fresenius Kabi AB, Uppsala, Sweden). In one case where theheart was procured as well, a Dacron Graft was sutured to the dividedpulmonary artery branches in order to reconstitute the integrity of thepulmonary artery (PA) and facilitate the connection of the lung to theEVLP circuit. The trachea was connected to the mechanical ventilator viaa silicon tube size matching the tracheal diameter. A temperature probewas positioned inside the left atrium. Initially, for de-airing thecircuit, the lungs were perfused at a flow rate of 0.5 L/min. The shuntfor de-airing on the inflow cannula was kept open until the organreached 32° C. and then closed for the rest of the session. The flow wasthen increased to 100% of estimated cardiac output for the specific setof lungs. The lungs were then warmed over 30 minutes to a target of 36°C. and the temperature difference between lung blood inflow and outflowwas not allowed to exceed 8° C. The flow rate was then increasedgradually to a target level of 70 mL/min per kilogram donor weight,during which the PA pressure was measured continuously and limited to 15mm Hg. Rewarming was achieved within 20-30 minutes. When the perfusatetemperature reached 32° C., mechanical ventilation was started involume-controlled mode at an initial tidal-volume of 3 ml per kilogramof donor weight with a positive end-expiratory pressure (PEEP) level of5 cm H₂O, a rate of 7-10 breaths/min, and a FiO₂ of 0.5. Tidal volumewas then increased gradually to a maximum of 7 mL per kilogram of donorweight. Perfusate samples for blood gas analyses were drawn from thededicated port of the system.

Cells

Human bone marrow derived MAPCs (Human MultiStem®, Athersys Inc.,Cleveland) were isolated from a single bone marrow aspirate, obtainedwith consent from a healthy donor, and processed according to previouslydescribed methods (Penn, M S et al., Circ Res 2012; 110(2):304-11;Maziarz, R T et al., Biology of Blood and Marrow Transplantation 2012;18(2 Sup):5264-5265; and clinicaltrials.gov #NCT01436487, #NCT01240915and #NCT01841632). In brief, MAPCs were cultured in fibronectin-coatedplastic tissue culture flasks under low oxygen tension in a humidifiedatmosphere of 5% CO₂. Cells were cultured in MAPC culture media(low-glucose DMEM [Life Technologies Invitrogen] supplemented with FBS(Atlas Biologicals, Fort Collins, Colo.), ITS liquid media supplement[Sigma], MCDB [Sigma], platelet-derived growth factor (R&D Systems,Minneapolis, Minn.), epidermal growth factor (R&D Systems),dexamethasone ([Sigma], penicillin/streptomycin [Life TechnologiesInvitrogen], 2-Phospho-L-ascorbic acid [Sigma, St. Louis, Mo.), andlinoleic acid-albumin (Sigma). Cells were passaged every 3-4 d,harvested using trypsin/EDTA (Life Technologies Invitrogen, Carlsbad,Calif.). The cells were positive for CD49c and CD90 and negative for MHCclass II and CD45 (all Abs were from BD Biosciences, Franklin Lakes,N.J.). Cells were subsequently frozen at population doubling 30-35 incryovials in the vapor phase of liquid nitrogen at a concentration of1-10×10⁶ in 1 ml (PlasmaLyte, 5% HSA and 10% DMSO). Immediately prior totheir use, MAPCs were thawed and used directly.

Cell Inoculations, Lung Incubations, and Bronchoalveolar Lavage (BAL)Fluid and Tissue Analyses

When the temperature as measured by the intra-atrial probe reachedapproximately 32° C., MultiStem 1 ml vials were thawed, diluted into 19ml of sterile saline and administered by bronchoscope into the proximalportion of the LLL bronchus. A similar volume of vehicle (20 ml ofsterile saline) was similarly inoculated into the proximal portion ofthe RLL bronchus. Five minutes after delivery of MultiStem, the lungswere connected to a Hamilton-C2 mechanical ventilator. After either 2 or4 hours of perfusion on the Vivoline system the experiments werestopped. Five minutes before stopping the perfusion, the samesubsegments of the RLL and LLL that had been previously inoculated witheither cells or vehicle were lavaged with 60 mL saline. The recoveredBAL fluid was then separated into aliquots of either raw BAL fluid forassessing total cell counts and cell differentials or was centrifuged(1200 g×10 min at 4° C.) and the supernatant was collected in separatetubes, snap frozen, and stored at −70° C. (Lathrop, M J et al., StemCells Translational Medicine (in press); and Goodwin, M. et al., StemCells 2011:29(7):1137-48). For one lung, BAL fluid samples were alsoobtained during rewarming phase before ventilation was started, justprior to MSC or vehicle delivery.

Total BAL fluid cell numbers were determined using an ADVIA® HematologyAnalyzer (Siemens Diagnostics, Johnson City, Tenn.). Cytospins were madeusing 5×10⁴ cells centrifuged onto pre-cleaned, pre-treated glass slides(Corning Incorporate, Corning, N.Y.) at 800 rpm for 8 min, driedovernight, and stained using DiffQuick (Hema 3 Stain Set, FisherScientific, Pittsburgh, Pa.). Cell populations were determined byblinded manual count of 200 cells performed by three separateindividuals (Lathrop, M J et al., Stem Cells Translational Medicine (inpress); and Goodwin, M. et al., Stem Cells 2011:29(7):1137-48). Proteincontent in undiluted BAL fluid was assessed by Bradford assay (Bio-Rad,Hercules, Calif.). The Human Cytokine Array Kit, Panel A (R&D Systems,Minneapolis, Minn.) was used to examine BAL fluid supernatants forsoluble cytokines, chemokines, and other substances including C5/Ca,CD40L, CD54, CXCL1, CXCL10, G-CSF, Gro-1α, IL-1α, IL-1β, IL-1RA, IL-6,IL-8, IL-10, IL-16, IL-23, IP-10, I-TAC, MCP-1, MIF, PAI-1, RANES,serpin E1, sICAM, sTREM-1, TNFα, and the relative amount of cytokinecompared to internal controls determined on a UVP Bioimaging system.(Upland, Calif.). Elisas for other specific cytokines were performedaccording to manufacturer's instructions, IL-10 (R&D Systems,Minneapolis, Minn., Cat#:D1000B), and STC1, TSG-6 and iNOS (MyBioSource,San Diego, Calif., Cat#s: MBS946255, MBS926793, MBS723617).

Histological Assessments

Following BAL at the end of the perfusion period, the lungs weresubsequently gravity fixed with 10% formalin at room temperature for 1hour. Fixed lungs were dissected and the areas where cells wereinstilled stored in 10% formalin prior to paraffin fixation. Mounted 5μm sections were then evaluated for histologic appearance. Lunginflammation was scored on 10 airways per animal, in a blinded fashionby three individuals, based on the presence and intensity ofperi-bronchial cell infiltrates compared to known positive and negativecontrols using an established semi-quantitative scoring system, using a0-3 range and 0.5 scale increments as previously described (Lathrop, M Jet al., Stem Cells Translational Medicine (in press); and Goodwin, M. etal., Stem Cells 2011:29(7):1137-48).

qPCR Analyses of Tissue Inflammatory Markers

Lung biopsy samples from lungs 2-5 were obtained using an automaticstapler (Covidien GIA™ DST Series™ 80 mm) from the periphery of the LLLand RLL just prior to cell or vehicle infusion at 2 and 4 hours aftercell or vehicle infusion and at the end of the experiment. The sampleswere snap frozen and subsequently homogenized and the expression levelsof inflammatory cytokine mRNAs determined by qPCR (see details below).

Samples were homogenized in RNA lysis buffer and RNA extracted using theRNeasy kit (Qiagen, Germantown, Md.) according to manufacturer'sinstructions. Additional DNase treatment was performed using theDNA-free kit (Life Technologies, Carlsbad, Calif.). RNA concentrationwas measured by NanoDrop 2000 (Thermo Scientific, Waltham, Mass.) and 1μg RNA was reverse transcribed using M-MLV Reverse Transcriptase(Promega, Madison, Wis.) followed by RNAse treatment using RNace-itCocktail (Agilent, Santa Clara, Calif.). Reverse transcriptase negativesamples and water were run as controls. 5 μl of the cDNA was mixed withSYBR green (Promega) and primers (IDT) and run on the ABI 7500 FASTsystem (Applied Biosystems, Foster City, Calif.). The samples werenormalized to GAPDH and expressed as a percent of Human Reference(Agilent)+/−standard deviation.

Primer sequences were as follows:

VEGFA-F1 (SEQ ID NO:1);

VEGFA-R1 (SEQ ID NO:2);

IGF1-F4 (SEQ ID NO:3);

IGF1-R4 (SEQ ID NO:4);

EGF-F1 (SEQ ID NO:5);

EGF-R1 (SEQ ID NO:6);

IL-10-F2 (SEQ ID NO:7);

IL-10-R2 (SEQ ID NO:8);

FGF2-F1 (SEQ ID NO:9);

FGF2-R1 (SEQ ID NO:10);

HGF-F1 (SEQ ID NO:11);

HGF-R1 (SEQ ID NO:12);

CCL5-F1 (SEQ ID NO:13);

CCL5-R1 (SEQ ID NO:14);

TGFB1-F1 (SEQ ID NO:15);

TGFB1-R1 (SEQ ID NO:16)

CXCL10-F1 (SEQ ID NO:17);

CXCL10-R1 (SEQ ID NO:18);

NOS3-F2 (SEQ ID NO:19);

NOS3-R2 (SEQ ID NO:20);

STC1-F1 (SEQ ID NO:21);

STC1-R1 (SEQ ID NO:22);

GAPDH-F1 (SEQ ID NO:23);

GAPDH-R1 (SEQ ID NO:24);

ANGPT1-F2 (SEQ ID NO:25);

ANGPT1-R2 (SEQ ID NO:26);

NOS2-F1 (SEQ ID NO:27);

NOS2-R1 (SEQ ID NO:28);

TNFAIP6-F1 (SEQ ID NO:29);

TNFAIP6-R1 (SEQ ID NO:30);

FGF7-F1 (SEQ ID NO:31); and

FGF7-R1 (SEQ ID NO:32).

Statistical Analysis

Groups were compared using either one way or two-way ANOVA with aFishers LSD post-test or by direct analysis between two groups byStudent's T-test, using a Welch's correction for unequal variances, asappropriate (Lathrop, M J et al., Stem Cells Translational Medicine (inpress); and Goodwin, M. et al., Stem Cells 2011: 29(7): 1137-48).

Results

The relevant clinical characteristics of the donor lungs are summarizedin Table 1.

TABLE 1 Clinical Characteristics of the Donor Lungs DonorCharacteristics 1 2 3 4 5 Mean ± SD Age 55 56 44 66 50 54.2 ± 3.6 SexMale Male Male Female Male Cause of Death CVA SH Asphyxiation IH MVAPaO₂ @100% FiO₂ 150  186  254  443  149  236.4 ± 55.1 Peep 10 10 10  510  9.0 ± 1.0 Radiographic Findings Infiltrate- Infiltrate-Infiltrate-Edema Clear Edema-Right Edema Edema lower lobe collapse,Right pleural effusion Lung Appearance Edematous Edematous EdematousMultiple Contusions, surface Edematous nodules CVA: Cerebrovascularaccident; SH: Subarachnoid hemorrhage; IH: Intracranial Hemorrhage; MVA:motor vehicle accident.

Donor age ranged from 44-66 and three of the five donor lungs wereobtained from patients with devastating neurologic events, one fromasphyxia, and one from a motor vehicle accident. Four of the five lungswere not deemed suitable for transplant because of poor functionalstatus including low PaO₂ values with a mean of 184.75 mmHg at 100% FiO₂at ±a PEEP of 10 mmHg. These lungs also had radiographic abnormalities,variously including contusions, significant emphysema, or lobar collapsethat did not respond to recruitment maneuvers in the operating room.Each of these lungs also had radiographic signs of pulmonary edema withtwo having also pleural effusion and all were noted to be variablyedematous following surgical removal. Lung #5 had RLL collapse on CXRbut expanded following removal and bronchoscopic removal of mucus plugs.One lung (lung #4) was physiologically suitable for donation with normalappearance, clear CXR, and good oxygenation on 5 mmHg PEEP but notutilized due to the presence of small surface nodules that weresubsequently found to be benign on biopsy.

A summary of the protocol utilized for each lung is presented in Table 2and also in schematic form in FIG. 1.

TABLE 2 Summary of Experimental Protocol Donor Lung 1 2 3 4 5 Mean ± SDDuration of 8 8 8 8 8 8.0 ± 0   Cold Static Storage (hours) Rewarming 2225 28 26 24 25.0 ± 1.0  Time (minutes) Duration of Ex 4 2.5 4 4 4 3.6 ±0.6 Vivo Perfusion (hours) Cells or Vehicle 10⁷ MSC to 10⁷ MSC to 10⁷MSC to LLL 10⁶ MSC to LLL 10⁷ MSC to LLL Delivered LLL LLL Vehicle toRLL Vehicle to RLL Vehicle to RLL Vehicle to RLL Vehicle to RLL

Overall the lungs had similar cold storage (8 hrs) and rewarming (25+2.2minutes) times and subsequently similar reperfusion times (3.7±0.6hours) following bronchoscopic administration of cells or vehicle. Atthe end of the reperfusion period, there was some degree of furtheredema that had developed in each lung and lung number 4 had also newlydeveloped some degree of edema. However, overall there was less visibleedema and inflammation in the MSC-treated (LLL) vs. vehicle-treated(RLL) lobes, even with the lower dose of MAPCs utilized in lung #4.Representative images are shown in FIG. 2.

Histologic assessment of the lungs at the end of the reperfusion perioddemonstrated that although patches of inflamed areas could be found insome of the MAPC-treated LLLs, there was significantly less overallinflammation in 4 out of the 5 lungs and also averaged over all 5 lungs,as assessed by semi-quantitative scoring of peribronchial, perivascular,and alveolar septa edema and by presence of inflammatory cellinfiltrates (FIG. 3). Representative photomicrographs are depicted inFIG. 4.

Total BAL fluid cell counts were obtained in two out of four lungs(lungs 3 and 5) receiving the higher cell dose. In both cases, there wasa significant decrease in the MSC-treated LLL compared to thevehicle-treated RLL (FIG. 5A). A trend towards decrease in total BALfluid cell counts was also observed in the lung receiving the lower MSCdose (lung 4, FIG. 5A). Cell differentials obtained on BAL fluid samplesfrom all five lungs demonstrated a consistent increase in neutrophilsand eosinophils in the vehicle-treated RLL that was ameliorated in theMSC-treated LLL (FIG. 5B). Measurements of BAL fluid total proteinlevels was variable between the lungs but a consistent decrease in totalprotein in the MSC-treated LLL vs. vehicle-treated RLL was observed inall 5 lungs (FIG. 5C).

An increase in levels of IL-10 in the MSC-treated LLL compared thevehicle treated RLL was observed (FIG. 6). However, other solubleanti-inflammatory mediators implicated in pre-clinical models of MSCactions in lung injury and other models, such as IL-1RA, STC, TGS-6, andiNOS, were not reliably increased in the MSC-treated LLL in any of the 5lungs (FIG. 6). Tissue mRNA levels were assessed in 4 of 5 lungs (lungs2-5) by qPCR analyses of biopsy samples obtained prior to cell orvehicle administration and then after either 2 or 4 hours of reperfusionperiod. Overall, patterns of tissue mRNA levels were more consistentbetween the 4 lungs. Comparable to BAL fluid levels of IL-10 protein,there was a 3.5-fold increase in the levels of tissue IL-10 mRNA in theMSC-treated LLL compared to only a 1.6-fold increase in vehicle-treatedRLL as assessed at 2 hrs (FIG. 7). Similar increases in LLL vs. RLL werealso observed at 2 hrs in mRNA levels of Angpt1 and STC1. Interestingly,for both the LLL and RLL there was a large increase in the foldexpression of TSG6 from 2 to 4 hours.

Discussion

A number of different methods have been studied to improve the viabilityof donor lungs and to decrease either warm or cold ischemic inflammatoryinjury. These include a flushing solution with extracellularcharacteristics delivered both in an antegrade and retrograde fashionand the use of a portable ex vivo preservation system currently underclinical investigation for use in transport of donor lungs (Machuca, T Net al., Surg Clin North Am 2013; 93(6):1373-94). Different areas ofresearch for therapeutic interventions aim to modulate the responseinduced by ischemia and reperfusion. For example experimental animalmodels have shown beneficial effect from gene therapy deliver of IL-10(Cypel, M. et al., Sci Transl Med. 2009; 1(4):4-9) and from adenosinereceptor activation (Fernandez, L G et al., J Thorac Cardiovasc Surg2013; 145(6):1654-9; and Mulloy, D P et al., Ann Thorac Surg 2013;95(5):1762-7). However, while the experimental data are promising, it isunlikely that modulating one out of many inflammatory pathways canregulate a phenomenon that alters several cellular mechanisms involved,as innate and adaptive immunity, the activation of the complementcascade, endothelial dysfunction, and the triggering of cell death. Incontrast, bone marrow-derived MSCs and MAPCs have the unique potentialof acting on multiple inflammatory pathways involved inischemia/reperfusion injury.

Ex vivo lung perfusion (EVLP) was originally designed as a method toassess the quality of lungs from donation after cardiac death (DCD) andfrom other non-acceptable donor lungs (Wierup, P. et al., Ann ThoracSurg 2006; 81(2):460-6; and Ingemansson, R. et al., Ann Thorac Surg2009; 87(1):255-60). This technique is currently under clinical trialfor the evaluation and reconditioning of potential donor lungs thatunder current criteria are not deemed suitable for transplant (Cypel, M.et al., N Engl J Med 2011; 364(15):1431-40). EVLP further offers anopportunity to administer MSCs or MAPCs directly into the donor lung byeither intratracheal or intravascular routes prior to implantation.Using this approach the inventors chose to initially assess directairway MAPC administration into a single lobe with the contralaterallung as comparison to directly assess effects within each individuallung. The cold ischemic storage (8 hours of total cold storage) wasprolonged beyond the actual times generally accepted for the lungs topotentiate any IRI that might develop and thus to maximize potentialanti-inflammatory actions of the MSCs. The inventors also chose to use“off the shelf” non-HLA matched MAPCs as proof of feasibility. Moreover,the inventors demonstrate a consistent and potent anti-inflammatoryeffect of the MAPCs. Notably, the change in cytokine profile,particularly increase in IL-10, may be particularly beneficial for IRI.

From the above description of the present invention, those skilled inthe art will perceive improvements, changes and modifications. Suchimprovements, changes, and modifications are within the skill of thosein the art and are intended to be covered by the appended claims. Allpatents, patent applications, and publications cited herein areincorporated by reference in their entirety.

What is claimed is:
 1. A method comprising transplanting an organ thathas been exposed to exogenous stem cells, wherein the stem cells arenon-embryonic, non-germ cells that express telomerase, have a normalkaryotype, are not tumorigenic, and wherein the stem cells haveundergone at least 10-40 cell doublings in cell culture prior to theirexposure to the organ, wherein the organ is exposed to the stem cellsprior to transplantation into a recipient.
 2. The method of claim 1,wherein the stem cells can differentiate into cell types of at least twoof endodermal, ectodermal, and mesodermal germ layers.
 3. The method ofclaim 1, wherein the stem cells express oct4.
 4. The method of claim 2,wherein the stem cells can differentiate into cell types of endodermal,ectodermal, and mesodermal germ layers.
 5. The method of claim 1,wherein the stem cells are non-embryonic, non-germ cells that expressoct4 and telomerase and can differentiate into cell types of at leasttwo of endodermal, ectodermal, and mesodermal germ layers.
 6. The methodof claim 5, wherein the stem cells can differentiate into cell types ofendodermal, ectodermal, and mesodermal germ layers.
 7. The method ofclaim 5 or 6, wherein the stem cells are non-HLA matched, allogeneiccells.
 8. The method of claim 1, wherein the stem cells have undergoneat least 30-35 cell doublings.
 9. The method of claim 1, wherein theconcentration of stem cells exposed to the organ is about 1×10⁶ cells/mlto about 10×10⁶ cells/ml.
 10. The method of claim 1, wherein the stemcells are exposed to the organ for about 2-4 hours.
 11. The method ofclaim 1, wherein the stem cells are contained in a fluid for perfusioninto the organ or in a carrier for intra-organ administration.
 12. Themethod of claim 1, wherein the stem cells are contained in a medium inwhich the organ is bathed prior to transplantation.
 13. The method ofclaim 1, wherein the organ is selected from the group consisting oflung, kidney, heart, liver, pancreas, thymus, gastrointestinal tract,and composite allografts.
 14. The method of claim 1, wherein exposure tothe stem cells reduces inflammation in the organ.
 15. The method ofclaim 1, wherein exposure to the stem cells reduces the occurrence ofinflammatory cells in the organ.
 16. The method of claim 1, whereinexposure to the stem cells reduces inflammatory cytokines in the organ.17. The method of claim 1, wherein exposure to the stem cells reducesischemic-reperfusion injury.
 18. The method of claim 1 wherein the stemcells are derived from human bone marrow.